Ovarian granulosa cells utilize scavenger receptor SR-BI to evade cellular cholesterol homeostatic control for steroid synthesis

Abstract

Cholesterol is an essential structural component of mammalian cell membrane and a precursor for the synthesis of steroid hormones and bile acids ( 1 ). During ovarian steroid hormone synthesis, cholesterol is fi rst transported into mitochondrial inner membrane facilitated by the steroidogenic acute regulatory protein (StAR), and then converted to the important sex steroid progesterone under sequential actions of the mitochondrial enzyme P450 cholesterol side chain-cleavage enzyme (P450scc) and endoplasmic reticulum enzyme 3 ␤ -hydroxysteroid dehydrogenase (3 ␤ -HSD). Progesterone could be further enzymatically processed into androgens and estrogens ( 2 ). Cellular cholesterol could be derived from the de novo synthesis pathway or from circulating lipoproteins. For steroidogenic cells, lipoproteins are the major source that provides suffi cient cholesterol to meet the demand of steroid hormone synthesis ( 3 ). Aside from the ubiquitous LDL receptor (LDLR)-mediated endocytic uptake of LDL-cholesterol adapted by most cell types, steroidogenic cells additionally utilize HDL-derived cholesterol through scavenger receptor class B member I Abstract Cellular cholesterol is known to be under homeostatic control in nonsteroidogenic cells, and this intrigued us to understand how such control works in ster oidogenic cells that additionally use cholesterol for steroid hormone synthesis. We employed primary culture of rat ovarian granulosa cells to study how steroidogenic cells adapt to acquire suffi cient cholesterol to meet the demand of active steroidogenesis under the stimulation of gonadotropin follicle-stimulating hormone (FSH) and cytokine transforming growth factor (TGF) ␤ 1. We found that TGF ␤ 1 potentiated FSH to upregulate scavenger receptor class B member I (SR-BI) and LDL receptor (LDLR), both functional in uptaking cholesterol as hHDL 3 NSC99-2320-B-010-013-MY3 (to J.-J.H.) and NSC98-2311-B-002-005-MY3 (to F.-C.K.) Abbreviations: 25-OHC, 25-hydroxycholesterol; 3 ␤ -HSD, 3 ␤ -hydroxysteroid dehydrogenase; ACTH, adrenocorticotropic hormone; AMG, aminoglutethimide; CE, cholesteryl ester; ChIP, chromatin immunoprecipitation; FSH, follicle-stimulating hormone; LDLR, LDL receptor; LRH-1, liver receptor homolog-1; P450scc, P450 cholesterol side chain-cleavage enzyme; SCAP, SREBP cleavage-activating protein; SR-BI, scavenger receptor class B member I; SRE, sterol response element; SREBP, sterol regulatory element-binding protein; StAR, steroidogenic acute regulatory protein; TGF ␤ 1, transforming growth factor ␤ 1. This study was supported by National Science Council of Taiwan Grants

Calcineurin and CRTC2 mediate FSH and TGF 1 upregulation of Cyp19a1 and Nr5a in ovary granulosa cells

“…cell lysates were prepared and subjected to chromatin immunoprecipitation (ChIP) analysis conducted as described previously (Lai et al 2013). In brief, 10% of each sample was saved as input, and the rest was immunoprecipitated with CRTC2, CREB, LRH1, or SF1 antibody, and isotypic normal IgG was used as a negative control.…”

“…At the end of culture, the cells were extracted for total RNA using TRIzol reagent (Life Technologies, Inc.), and then RNA sample was analyzed using RT-PCR and agarose gel electrophoresis conducted as described previously (Lai et al 2013) for Cyp19a1, Nr5a1, and Nr5a2, with Gapdh used as an internal control. Forward and reverse primer pairs used are listed in Table 1.…”

“…Immunoblotting was carried out as previously described (Lai et al 2013). An equal amount of protein was loaded for each sample, which was then analyzed for aromatase, LRH1, and SF1, with b-actin used as an internal control.…”

CRTC2 and Nedd4 ligase involvement in FSH and TGFβ1 upregulation of connexin43 gap junction

“…Granulosa cells were treated as described below and in the figure legends. This granulosa cell model does not undergo spontaneous luteinization as control cells under cultivation period remain flattened and secrete very low levels of progesterone (see Fang et al 2012, Lai et al 2013 and is ideal for studying the regulation of granulosa cell differentiation.…”

“…At the end of culture, cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS and permeabilized with 0.05% Triton X-100 for 5 min, washed again and then blocked in 3% BSA and 3% goat serum/PBS for 1 h. Cells were then incubated with rabbit polyclonal antibody against Cx43 (Sigma) (Boswell et al 2010) or isotypic IgG serving as a negative control for 1 h, followed by FITC-conjugated goat anti-mouse IgG for 1 h. The cover slips were then mounted on glass slides and viewed under a fluorescent microscope (Olympus BX50, Tokyo, Japan) equipped with a mercury arc lamp and photographed using SPOT image capture system (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Precaution was taken to have fluorescent images reflecting the actual emission signal for comparison among samples as previously described (Lai et al 2013).…”